UBC Theses and Dissertations
Crystallographic studies of bacterial proteins involved in sialic acid synthesis Gunawan, Jason
In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion, and are synthesized by a conserved enzymatic pathway which includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetyl mannosamine (ManNAc), directly forming N-acetylneuraminic acid (NeuNAc or sialic acid). Despite the important biological roles of sialic acids, very little is known about the structural and mechanistic properties of the enzymes which create them. This thesis primarily focuses on the determination of the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (β/α)₈ barrel (TIM barrel) type fold at the N-terminal end, and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. The structures of NeuB in the malate-bound form, bound to PEP and the substrate analog N-acetylmannosaminitol, and bound to the intermediate analogue N-acetylneuraminc borate were determined to 1.9, 2.2, and 2.2 Å resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature which leads to distinct modes of substrate binding from other PEP utilizing enzymes which lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered Mn²⁺ and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the ManNAc carbonyl to the addition of PEP. In addition, the development of a coupled assay to monitor NeuB reaction kinetics, and an ¹⁸O-labelling study that demonstrates the synthase operates via a C-O bond cleavage mechanism are discussed in order to further analyze structure-function relationships.
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